1. Volume 62, Issue 6, Pages 862-874 (June 2016)
Taz1-Shelterin Promotes Facultative Heterochromatin Assembly at Chromosome- Internal Sites Containing Late Replication Origins 
Martin Zofall, Deborah R. Smith, Takeshi Mizuguchi, Jothy Dhakshnamoorthy, Shiv I.S. Grewal 
Molecular Cell 
Volume 62, Issue 6, Pages (June 2016)
DOI: /j.molcel
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2. Molecular Cell 2016 62, 862-874DOI: (10.1016/j.molcel.2016.04.034)
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3. Figure 1
Genetic Screen for Mutations that Specifically Compromise Silencing at a Non-DSR Heterochromatin Island
(A) Genome-wide distribution of non-DSR and DSR/MTREC-dependent heterochromatin islands. Peaks of H3K9me2 along S. pombe chromosomes are shown. Asterisks denote non-DSR islands. Data from Zofall et al. (2012) was used for this analysis.
(B) The strategy used to isolate mutants specifically defective in silencing at island 3.
(C) Serial dilution analyses to assay expression of Is3::ura4+ or mat2::ade6+. Wild-type and mutant strains were spotted on the indicated medium. Red coloration on adenine-limited media indicates ade6+ repression, while white color indicates expression.
(D) Heterochromatin loss at island 3 in mut-5. H3K9me2 enrichment was determined by duplex PCR of DNA isolated from immunoprecipitated chromatin or from whole-cell extracts (WCEs). Enrichments were calculated as the ratio of band intensities observed in the ChIP sample PCR normalized to the ratio in the input (WCE) PCR. Average enrichments (top number) and SD of three measurements (bottom number) are indicated.
(E) H3K9me2 distribution at individual loci in the indicated strains, as determined by ChIP-chip. Note that mut-5 cells lack heterochromatin at non-DSR islands (3 and 15), whereas heterochromatin is unaffected at DSR-dependent islands (1 and 9). Chromosome positions in (A) and (E) correspond to the Sanger Center S. pombe database 2007 assembly.
See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
The mut-5 Phenotype Is Associated with a Mutation in the DNA-Binding Domain of the Telomere-Associated Protein Taz1
(A) Identification of the mut-5 mutation by genome sequencing. The mut-5 phenotype was tracked through a series of backcrosses by assaying for FOA sensitivity of mut-5 Is3::ura4+ cells.
(B) The domain structure of Taz1 and its human homolog is indicated together with the sequence substitution found in mut-5 cells.
(C and D) The loss of island 3 silencing in mut-5 is phenocopied by taz1Δ. (C) Serial dilutions of the indicated strains on complete media, counter-selective (+FOA) media, and media lacking uracil. (D) ChIP of H3K9me2 at island 3 in the indicated strains. Quantitative duplex PCR was used to compare the relative enrichments of island 3 DNA in H3K9me2-immunoprecipitated and input samples. The average enrichments and standard deviations of three measurements are indicated between the panels.
See also Figure S2.
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5. Figure 3 Taz1 Associates with Taz1-Dependent Heterochromatin Islands
(A) Distribution of H3K9me2 and Taz1-GFP along S. pombe chromosomes as determined by ChIP-chip. Islands affected in mut-5 cells are labeled in red. H3K9me2 data from Zofall et al. (2012) was used.
(B and C) Taz1 preferentially localizes to non-DSR islands containing telomeric repeat sequences. Note that island 7 contains a putative variant binding sequence. (B) Taz1-GFP distribution at non-DSR islands as determined by ChIP-chip. ORFs, the positions of Taz1-binding sites and late replicating origins are indicated. (C) Conventional ChIP of Taz1-GFP at the indicated islands. The average enrichments and standard deviations of three measurements are indicated.
(D) Taz1-GFP distribution at DSR islands.
See also Figure S3.
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6. Figure 4
Taz1 Localization to Taz1-Dependent Islands Is Required for Heterochromatin Assembly
(A) Genome-wide distribution of H3K9me2 in wild-type and taz1Δ cells as determined by ChIP-chip. Taz1-bound islands are labeled in red.
(B) H3K9me2 distribution at Taz1-bound islands in wild-type and taz1Δ cells. Open reading frame (ORF) positions are indicated above the panels, and late replication origins are marked by red ovals.
Wild-type H3K9me2 microarray data used in (A) and (B) are from Zofall et al. (2012).
(C) Conventional ChIP at selected islands. Duplex PCR was performed using DNA isolated from H3K9me2 immunoprecipitated fractions and input DNA from whole-cell extracts. The average relative enrichments and SDs of replicates are shown.
(D and E) Internal telomere repeat-like sequences are required for the assembly of non-DSR islands 3 and 15. (D) Conventional ChIP of Taz1-GFP in wild-type and strains containing deletions of the Taz1-binding sites at island 3 (Is3telrΔ) or island 15 (Is15telrΔ). (E) Conventional ChIP of H3K9me2 at islands 3 and 15 in the indicated strains. The deleted Taz1-binding motifs are indicated. The entire deleted sequence for each island can be found in Table S3.
See also Figure S4.
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7. Figure 5
Taz1 and Heterochromatin Factors Affect Gene Repression at Taz1-Dependent Islands
(A) mRNA levels at the indicated Taz1-dependent islands were determined by strand-specific RT-PCR. The locations of the Taz1-binding sequences and the RT-PCR-amplified cDNA fragments (red bars) are indicated.
(B and C) Taz1 is not required for gene repression at Taz1-independent heterochromatin sites. (B) mRNA levels at DSR heterochromatin islands in wild-type, taz1Δ, and red1Δ. Note the lack of derepression at DSR-containing islands 6 and 9 targeting mei4 and ssm4 loci in taz1Δ. (C) Heterochromatin-mediated repression of pericentromeric dg/dh repeats is preserved in taz1Δ. RT-PCR of dh repeat RNAs. act1 mRNA is used as a loading control.
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8. Figure 6
Heterochromatin Factors Affect the Timing of Replication of Late Origins at Heterochromatin Islands
Taz1 and heterochromatin factor Clr4 delay replication at a telomere region (first panel from left) and Taz1-dependent islands. ChIP-chip analysis of BrdU incorporation in early S phase cells is shown. Cells carrying the cdc25-22 allele were arrested at the G2/M boundary, supplemented with HU and BrdU, and released from the block by a temperature shift. The BrdU-labeled DNA was immunoprecipitated from early S phase cells and quantified using microarray analyses. The difference between mutant and wild-type cells is plotted. The distribution of Taz1-GFP and H3K9me2 is also shown. The positions of ORFs and replication origins (red ovals) are indicated above the charts. H3K9me2 microarray data from Zofall et al. (2012) was used. See also Figure S5.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7
Shelterin Components and Rif1 Are Required for H3K9me at Taz1-Dependent Islands
(A) Conventional ChIP combined with duplex PCR was used to assay H3K9me2 at islands 3 and 14. The average enrichments and SDs are indicated.
(B) Shelterin components affect gene repression at Taz1 targeted islands. RT-PCR was used to examine mRNA levels at Taz1-dependent heterochromatin islands in the indicated strains. act1 mRNA was used as a loading control.
(C) The Shelterin component Ccq1 associates with the Clr4 H3K9 methyltransferase, as determined by immunoprecipitation and western blot analysis. The FLAG-tagged Clr4 co-purifying fraction was probed for Ccq1 and Clr4.
(D) Model showing the involvement of Shelterin components and Rif1 in the assembly of facultative heterochromatin islands targeting late origins. Taz1 bound to DNA and other Shelterin subunits, including Ccq1, assemble facultative heterochromatin islands to regulate gene expression and control replication timing. The control of replication timing at Taz1-dependent islands also involves Rif1 (Hayano et al., 2012; Tazumi et al., 2012). Taz1 might also cooperate with additional factors, such as HDAC, to directly affect replication initiation.
See also Figure S6.
Molecular Cell  , DOI: ( /j.molcel )
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